The invention relates to the nucleotide and amino acid sequences and to the activity and use of the fluorescent protein CGFP....http://www.google.es/patents/US7879557?utm_source=gb-gplus-sharePatente US7879557 - Isolated fluorescent protein from Clytia gregaria CGFP and use thereof

5. A method of determining whether a gene of interest, or fragment thereof, has been expressed comprising monitoring the fluorescence of a polypeptide encoded by a fusion gene and comparing it to the fluorescence when the gene or fragment is not expressed, wherein said fusion gene comprises the nucleic acid of claim 1 operably linked to said gene of interest, or fragment thereof.

6. The recombinant vector of claim 3, wherein the vector is an expression vector.

7. The vector of claim 6, wherein said vector comprises an inducible promoter.

8. A method of producing a fluorescent protein encoded by the nucleic acid of claim 1 in a host cell, wherein said host cell is a bacteria cell or a eukaryotic cell comprising the steps of:

(i) transforming said host cell with the expression vector of claim 6, and

(ii) growing said host cell from step (i) under conditions that permit said fluorescent polypeptide to be produced in the transformed host cell of part (i).

9. An isolated nucleic acid molecule, selected from the group consisting of:

c) a nucleic acid molecule which is at least 95% homologous to the full-length nucleic acid sequence SEQ ID NO:1 and whose complementary strand hybridizes under stringent conditions with a nucleic acid molecule encoding the amino acid sequence of SEQ ID NO: 2 or with a nucleic acid molecule consisting of the nucleic acid sequence of SEQ ID NO: 1, and which encodes a fluorescent protein having an excitation peak of about 475 nm and an emission peak of about 493 nm.

10. The method of claim 8, further comprising isolating the fluorescent polypeptide.

Descripción

The invention relates to the nucleotide and amino acid sequences and to the activity and use of the fluorescent protein CGFP (fluorescence protein of Clytia gregaria).

The fluorescent proteins differ not only due to their nucleotide and amino acid sequences but also due to their biochemical and physical properties. The spectral characteristics of the fluorescent proteins may differ both on the side of excitation and on, the side of emission. An overview of the fluorescence spectra and the excitation wavelength can be found in Table 2.

TABLE 2

Overview over some fluorescent proteins. Indicated are the organism

from which the protein has been isolated, the excitation wavelength

and emission wavelength determined in spectral analyses.

Organism

Excitation

Fluorescence

Aequoria

465-498

nm

509 nm

Halistaura

465

nm

497 nm

Phialidium

485

nm

498 nm

Renilla

498

nm

508 nm

The use of fluorescent proteins has already been described previously. An overview can be found in Table 3:

TABLE 3

Overview over some fluorescent proteins. Indicated are the organism

from which the protein has been isolated, the name of the fluorescent

protein and a selection of patents or applications.

Organism

Fluorescent protein

Patent/application

Renilla mulleri

GFP

U.S. Pat. No. 6,436,682

WO200168824

WO200257451

WO200134824

WO9949019

U.S. Pat. No. 6,232,107

Aequoria

GFP

WO200071565

WO9711094

WO9623898

U.S. Pat. No. 5,958,713

U.S. Pat. No. 6,172,188

It was shown that it is possible to alter the physical and biochemical properties of fluorescent proteins by altering the amino acid sequence thereof. Examples of mutagenized fluorescent proteins have been described in the literature (Delagrave et al., 1995; Ehrig et al., 1995; Heim et al., 1996).

Fluorescent proteins are already used in a wide variety of areas. The use of fluorescent proteins in ‘Fluorescence Resonance Energy Transfer’ (FRET), ‘Bioluminescence Resonance Energy Transfer (BRET) and other energy transfer methods has already been described in the literature (Mitra et al., 1996; Ward et al., 1978; Cardullo et al, 1988; U.S. Pat. Nos. 4,777,128; 5,126,508; 4,927,923; 5,279,943). Further nonradioactive methods of energy transfer by means of GFP have likewise been described previously (PCT appl. WO 98/02571 and WO 97/28261)

Reporter Systems

Reporter gene or indicator gene generally refers to genes whose gene products can be readily detected with the aid of simple biochemical or histochemical methods. At least 2 types of reporter genes are distinguished.

1. Resistance genes. Resistance genes refer to genes whose expression imparts to a cell the resistance to antibiotics or other substances whose presence in the growth medium leads to cell death when the resistance gene is absent.

Luminescence refers to the emission of photons in the visible spectral range, said emission being effected by excited emitter molecules. In contrast to fluorescence, the energy is supplied here not from the outside in the form of radiation of shorter wavelength.

A distinction is made between chemiluminescence and bioluminescence. Chemiluminescence refers to a chemical reaction resulting in an excited molecule which luminesces itself when the excited electrons return to the ground state. If this reaction is catalysed by an enzyme, then this process is called bioluminescence. The enzymes involved in the reaction are generally referred to as luciferases.

Classification of the Species Clytia gregaria

Cnidaria→Leptomedusae→Campanulariidae→Clytia gregaria

The species Clytia gregaria belongs to the cnidaria, especially to the medusae. The bioluminescent or fluorescent phenotype has already been described in 1998 (Ward et al., 1998).

Isolation of cDNA

In order to study the fluorescent activity of the species Clytia gregaria, specimens were caught in Friday Harbor in Washington State (USA) and stored in liquid nitrogen. The cDNA library was prepared using exclusively the bioluminescent ring of one medusa specimen. The Clytia gregaria cDNA libraries were generated by isolating the RNA by means of isothiocyanate according to the method of Krieg (Krieg et al., 1996).

The cDNA was prepared by carrying out an RT-PCR. To this end, 10 μg of RNA were incubated with reverse transcriptase (Superscribt Gold II) according to the following plan:

PCR

1.

30

seconds

95° C.

2.

6

minutes

68° C.

3.

10

seconds

95° C.

4.

6

minutes

68° C.

17 cycles of step 4 after step 3

In order to inactivate the polymerase, the reaction products were incubated at 37° C. with proteinase K for 30 minutes and cDNA was precipitated with ethanol. The cDNA was dissolved in water and incubated with SfiI at 37° C. for one hour. The reaction products were gel-filtrated in order to remove small fragments. The fractionated cDNA was then ligated into the SfiI-cut and dephosphorylated λTriplEx2 vector. A λ-phage expression library was then prepared by packing the cloned cDNA fragments into λ phages by means of the in vitro packing system SMART cDNA Library Construction Kits (Clontech).

The recombinant phages containing a cDNA insertion with potential expression of fluorescent proteins were identified by carrying out a “library screening”.

To this end, bacterial lawns of transformed E. coli XL1-Blue were plated on 90 mm culture dishes and incubated at 31° C. for 12-15 hours. Induction of protein expression was started by adding to the plates 60 μl of a 20 mM IPTG (isopropylthiogalactoside) solution. After 24 hours of incubation at room temperature, the plates were stored at 4° C. for 72 hours. This was followed by measuring the fluorescence.

To this end, the bacteria were irradiated using an argon laser (LGN502) with 100 mV at 488 nm or 366 nm (UVL21). The fluorescence was measured using a 510 nm ZSV filter.

To isolate the clones and for spectral analysis, the biomass of fluorescence-positive clones was removed from the culture plates and resuspended in PBS (phosphate buffered saline). The cells were disrupted by means of ultrasound. After clarifying the lysate by centrifugation, the fluorescence of the supernatant was determined in a fluorimeter.

A fluorescent protein was identified. The fluorescent protein was referred to as CGFP (fluorescence protein of clytia gregaria). The fluorescent protein CGFP is illustrated in detail below.

CGFP

The fluorescent protein CGFP shows the highest homology at the amino acid level to Aequoria GFP with an identity of 44% (depicted in Example 8; FIG. 5). At the nucleic acid level, the identity is below 30% (depicted in Example 9; FIG. 6). The BLAST method was used for sequence comparison (Altschul et al., 1997).

The invention also relates to functional equivalents of CGFP. Functional equivalents are those proteins which have comparable physicochemical properties and which are at least 70% homologous. Preference is given to a homology of 80% or 90%. Particular preference is given to a homology of 95%.

The fluorescent protein CGFP is suitable as a reporter gene for cellular systems, especially for receptors, for ion channels, for transporters, for transcription factors or for inducible systems.

The fluorescent protein CGFP is suitable as a reporter gene in bacterial and eukaryotic systems, especially in mammalian cells, in bacteria, in yeasts, in baculo, in plants.

The fluorescent protein CGFP is suitable as a reporter gene for cellular systems in combination with bioluminescent or chemiluminescent systems, especially systems with luciferases, with oxygenases, with phosphatases.

The fluorescient protein CGFP is suitable as a fusion protein, especially for receptors, for ion channels, for transporters, for transcription factors, for proteinases, for kinases, for phosphodiesterases, for hydrolases, for peptidases, for transferases, for membrane proteins, for glycoproteins.

The fluorescent protein CGFP is suitable for immobilization, especially by antibodies, by biotin, by magnetic or magnetizable carriers.

The fluorescent protein CGFP is suitable as a label of substrates or ligands, especially for proteases, for kinases, for transferases.

The fluorescent protein CGFP is suitable for expression in bacterial systems, especially for titer determination, as substrates for biochemical systems, especially for proteinases and kinases.

The fluorescent protein CGFP is suitable as a marker, especially coupled to antibodies, coupled to enzymes, coupled to receptors, coupled to ion channels and other proteins.

The fluorescent protein CGFP is suitable as a reporter gene in pharmacological drug screening, especially in HTS (High Throughput Screening).

The fluorescent protein CGFP is suitable as a component of detection systems, especially for ELISA (enzyme-linked immunosorbent assay), for immunohistochemistry, for Western blotting, for confocal microscopy.

The fluorescent protein CGFP is suitable as a marker for the analysis of interactions, especially for protein-protein interactions, for DNA-protein interactions, for DNA-RNA interactions, for RNA-RNA interactions, for RNA-protein interactions (DNA deoxyribonucleic acid; RNA: ribonucleic acid).

The fluorescent protein CGFP is suitable as a marker or fusion protein for expression in transgenic organisms, especially in mice, in rats, in hamsters and other mammals, in primates, in fish, in worms, in plants.

The fluorescent protein CGFP is suitable as a marker or fusion protein for analysing embryonic development.

The fluorescent protein CGFP is suitable as a reporter coupled to nucleic acids, especially to DNA, to RNA.

The fluorescent protein CGFP is suitable as a reporter coupled to proteins or peptides.

The fluorescent protein CGFP coupled to nucleic acids or peptides is suitable as a probe, especially for Northern blots, for Southern blots, for Western blots, for ELISA, for nucleic acid sequencings, for protein analyses, chip analyses.

The fluorescent protein CGFP is suitable as a label of pharmacological formulations, especially of infectious agents, of antibodies, of small molecules.

The fluorescent protein CGFP is suitable for geological studies, especially for sea currents, groundwater currents and river currents.

The fluorescent protein CGFP is suitable for expression in expression systems, especially in in-vitro translation systems, in bacterial systems, in yeast systems, in baculo systems, in viral systems, in eukaryotic systems.

The fluorescent protein CGFP is suitable for visualizing tissues or cells in surgical procedures, especially in invasive, in noninvasive, in minimally invasive procedures.

The fluorescent protein CGFP is also suitable for labeling tumour tissues and other phenotypically altered tissues, especially in histological examination, in surgical interventions.

The invention also relates to purifying the fluorescent protein CGFP, especially as wild-type protein, as fusion protein, as mutagenized protein.

The invention also relates to the use of the fluorescent protein CGFP in the field of cosmetics, especially of bath additives, of lotions, of soaps, of body paints, of toothpaste, of body powders.

The invention also relates to the use of the fluorescent protein CGFP for colouring, especially of food, of bath additives, of ink, of textiles, of plastics.

The invention also relates to the use of the fluorescent protein CGFP for the colouring of paper, especially of greeting cards, of paper products, of wallpapers, of handicraft articles.

The invention also relates to the use of the fluorescent protein CGFP for the colouring of liquids, especially for water pistols, for fountains, for beverages, for ice cream.

The invention also relates to the use of the fluorescent protein CGFP for producing toys, especially finger paints, face paints.

The invention relates furthermore to nucleic acid molecules, selected from the group consisting of

c) nucleic acid molecules whose complementary strand hybridizes under stringent conditions with a nucleic acid molecule of a) or b) and which have the biological function of a fluorescent protein;

d) nucleic acid molecules which differ from those mentioned under c) due to the degeneracy of the genetic code;

e) nucleic acid molecules whose sequences are at least 95% homologous to SEQ ID NO: 1 and which have the biological function of a fluorescent protein; and

f) nucleic acid molecules whose sequences are at least 65% homologous to SEQ ID NO: 1 and which have the biological function of a fluorescent protein.

The invention relates to the abovementioned nucleic acid molecules whose sequence contains a functional promoter 5′ of the sequence.

The invention also relates to nucleic acid molecules as described above which are a part of recombinant DNA or RNA vectors.

The invention relates to organisms which contain such a vector.

The invention refers to oligonucleotides, having more than 10 contiguous nucleotides which are identical or complementary to the DNA or RNA sequence of the CGFP molecules.

The invention relates to fluorescent proteins which are encoded by the above-described nucleotide sequences.

The invention refers to methods of expressing the fluorescent polypeptides according to the invention in bacteria, eukaryotic cells or in in vitro expression systems.

The invention also relates to methods of purifying/isolating a fluorescent polypeptide according to the invention.

The invention relates to peptides, having more than 5 contiguous amino acids which are recognized immunologically by antibodies to the fluorescent proteins according to the invention.

The invention relates to the use of the fluorescent proteins according to the invention as marker genes and reporter genes, in particular for pharmacological drug screening and diagnostics.

Expression of the Fluorescent Proteins According to the Invention

Expression refers to the production of a molecule which, after introduction of the gene into a suitable host cell, allows the foreign gene cloned into an expression vector to be transcribed and translated. Expression vectors contain the control signals required for expression of genes in cells of prokaryotes or eukaryotes.

Expression vectors may be constructed in principle in two different ways. In the case of “transcription fusions”, the protein encoded by the cloned-in foreign gene is synthesized as an authentic, biologically active protein. To this end, the expression vector carries all 5′ and 3′ control signals required for expression.

In the case of “translation fusions”, the protein encoded by the cloned-in foreign gene is expressed as a hybrid protein together with another protein which can be readily detected. The 5′ and 3′ control signals required for expression, including the start codon and possibly part of the sequences coding for the N-terminal regions of the hybrid protein to be formed, are derived from the vector. The additional protein part introduced not only stabilizes, in many cases, the protein encoded by the cloned-in foreign gene against degradation by cellular proteases but can also be used for detecting and isolating the hybrid protein formed. Expression may be either transient or stable. Suitable host organisms are bacteria, yeasts, viruses and eukaryotic systems.

Purification of the Fluorescent Proteins According to the Invention

The isolation of proteins (also after overexpression) is frequently referred to as protein purification. A multiplicity of established methods and processes are available for protein purification.

Solid-liquid separation is a basic operation in protein isolation procedures. This process step is required both in the removal of cells from the culture medium and in clarifying the crude extract, after cell disruption and removal of the cell debris, in removing precipitates after precipitations, etc. It is carried out by way of centrifugation and filtration.

By obtaining intracellular proteins requires the cell wall to be destroyed or made permeable. Depending on the scale and organism, high-pressure homogenizers or stirred ball mills or glass bead mills are used for this purpose. On the laboratory scale, mechanical cell integration and ultrasound treatment are used inter alia.

Various precipitation methods involving salts (in particular ammonium sulphate) or organic solvents (alcohols, acetone) are a rapid and deficient method of concentrating proteins, both for extracellular and intracellular proteins (after cell disruption). When purifying intracellular proteins, removal of the soluble nucleic acids is desirable (precipitation with streptomycin or protamine sulphate, for example). The recovery of extracellular proteins frequently involves the addition of carriers (e.g. starch, kieselguhr), prior to addition of the precipitants, in order to obtain better manageable precipitates.

Numerous chromatographic and partition methods are available for fine purification (absorption chromatography and ion exchange chromatography, gel filtration, affinity chromatography, electrophoreses). Column chromatography is also applied on the industrial scale. Affinity chromatography which makes purification factors of up to several 100 per step possible is especially important on the laboratory scale.

Extracellular proteins are obtained in relatively diluted solutions. Like extracellular proteins, they must be concentrated prior to further use. Ultrafiltration is a proven method, also on the industrial scale, in addition to the methods already mentioned.

Inorganic salts are frequently undesired accompanying substances of proteins in specific applications. They may be removed, inter alia, by gel filtration, dialysis and diafiltration.

Numerous proteins are used as dry preparations. Important drying processes are vacuum drying, freeze drying and spray drying.

Nucleotide and Amino Acid Sequences

The fluorescent protein CGFP is encoded by the following nucleotide sequence (SEQ ID NO: 1):

5′-

atgactgcacttaccgaaggagcaaaactgttcgagaaagaaattccctacattaca

gagttggaaggagacgttgaaggaatgaaattcatcatcaaaggtgaaggtactggc

gacgctactactggcaccatcaaagcgaaatatatttgcacaactggtgaccttcct

gtaccatgggctaccatcttgagtagtttgtcgtatggtgttttctgtttcgctaag

tatccacgccacattgccgactttttcaagagcacacaaccagatggttattcacaa

gacagaatcattagttttgacaatgatggacaatacgatgtcaaagccaaggttact

tatgaaaacggaacactttataatagagtcacagtcaaaggtactggcttcaaatca

aacggcaacatccttggtatgagagttctctaccattcaccaccacacgctgtctac

atccttcctgaccgtaaaaatggtggcatgaaaattgaatacaataaggctttcgac

gttatgggcggtggtcaccaaatggcgcgtcacgcccaattcaataaaccactagga

gcctgggaagaagattatccgttgtatcatcatcttaccgtatggacttctttcgga

aaagatccggatgatgatgaaactgaccatttgaccatcgtcgaagtcatcaaagct

gttgatttggaaacataccgttga-3′.

This results in an amino acid sequence of (SEQ ID NO: 2):

MTALTEGAKLFEKEIPYITELEGDVEGMKFIIKGEGTGDATTGTIKAKYICTTGDLP

VPWATILSSLSYGVFCFAKYPRHIADFFKSTQPDGYSQDRIISFDNDGQYDVKAKVT

YENGTLYNRVTVKGTGFKSNGNILGMRVLYHSPPHAVYILPDRKNGGMKIEYNKAFD

VMGGGHQMARHAQFNKPLGAWEEDYPLYHHLTVWTSFGKDPDDDETDHLTIVEVIKA

VDLETYR

These sequences can be found in the sequence listing.

DESCRIPTION OF THE FIGURES

FIG. 1 depicts the plasmid map of the pTriplEX2-CGFP vector.

FIG. 2 depicts the plasmid map of the pcDNA3-CGFP vector.

FIG. 3 depicts the transient expression of CGFP in CHO cells in the pcDNA3-CGFP expression vector. The figure depicts the microscopic image of the transiently transfected cells, at an excitation of 480 nm and an emission of 520 nm.

The vector used for preparing the construct illustrated below was the plasmid pTriplEx2 from Clontech. The derivative of said vector was referred to as pTriplEx2-CGFP. The pTriplEx2-CGFP vector was used for expressing CGFP in bacterial systems.

FIG. 1 depicts the plasmid map of the pTriplEX2-CGFP vector.

Example 2

The vector used for preparing the construct illustrated below was the plasmid pcDNA3.1(+) from Clontech. The derivative of said vector was referred to as pcDNA3-CGFP. The pcDNA3-CGFP vector was used for expressing CGFP in eukaryotic systems.

FIG. 2 depicts the plasmid map of the pcDNA3-CGFP vector.

Example 3

Bacterial Expression

Bacterial expression was carried out in the E. coli strain BL21(DE3) by transforming the bacteria with the expression plasmids pTriplEX2-CGFP and pTriplEX2. The transformed bacteria were incubated in LB medium at 37° C. for 3 hours and expression was induced for 4 hours by adding IPTG up to a final concentration of 1 mM. The induced bacteria were harvested by centrifugation, resuspended in PBS and sonicated. The fluorescence was determined with the aid of a fluorimeter.

Example 4

Eukaryotic Expression

Constitutive eukaryotic expression was carried out in CHO cells by transfecting said cells with the expression plasmids pcDNA3-CGFP and pcDNA3.1(+) in transient experiments. To this end, 10000 cells per well were plated in DMEM-F12 medium on 96-well microtiter plates and incubated at 37° C. overnight. Transfection was carried out with the aid of the Fugene 6 kit (Roche) according to the manufacturer's information. The transfected cells were incubated in DMEM-F12 medium at 37° C. overnight. The fluorescence was measured in a fluorimeter at room temperature.

FIG. 3 depicts expression of CGFP in CHO cells.

Example 5

Spectrum of the Fluorescent Protein CGFP

To measure the emission spectrum, E. coli BL21(DE3) were transformed with the plasmids pTriplEX2-CGFP and pTriplEX2. Induction was carried out by adding 1 mM IPTG and incubating at 37° C. for 4 hours. The bacteria were subsequently harvested and resuspended in PBS. The lysis was carried out using ultrasound. Subsequently, fluorescence was measured in a fluorimeter.